Reduced Expression of a Novel Midgut Trypsin Gene Involved in Protoxin Activation Correlates with Cry1Ac Resistance in a Laboratory-Selected Strain of Plutella xylostella (L.).

Shaoli Wang,Zhaojiang Guo,Lijun Gong,Youjun Zhang,Mazarin Akami,Dan Sun,Alejandra Bravo,Junlei Zhou,Mario Soberón,Qingjun Wu,Yang Bai,Lizhang Wen,Jianying Qin,Zhongxia Yang,Le Guo,Liuhong Zhu,Shi Kang,Fan Ye,Baoyun Xu

doi:10.3390/toxins12020076

Abstract

Bacillus thuringiensis (Bt) produce diverse insecticidal proteins to kill insect pests. Nevertheless, evolution of resistance to Bt toxins hampers the sustainable use of this technology. Previously, we identified down-regulation of a trypsin-like serine protease gene PxTryp_SPc1 in the midgut transcriptome and RNA-Seq data of a laboratory-selected Cry1Ac-resistant Plutella xylostella strain, SZ-R. We show here that reduced PxTryp_SPc1 expression significantly reduced caseinolytic and trypsin protease activities affecting Cry1Ac protoxin activation, thereby conferring higher resistance to Cry1Ac protoxin than activated toxin in SZ-R strain. Herein, the full-length cDNA sequence of PxTryp_SPc1 gene was cloned, and we found that it was mainly expressed in midgut tissue in all larval instars. Subsequently, we confirmed that the PxTryp_SPc1 gene was significantly decreased in SZ-R larval midgut and was further reduced when selected with high dose of Cry1Ac protoxin. Moreover, down-regulation of the PxTryp_SPc1 gene was genetically linked to resistance to Cry1Ac in the SZ-R strain. Finally, RNAi-mediated silencing of PxTryp_SPc1 gene expression decreased larval susceptibility to Cry1Ac protoxin in the susceptible DBM1Ac-S strain, supporting that low expression of PxTryp_SPc1 gene is involved in Cry1Ac resistance in P. xylostella. These findings contribute to understanding the role of midgut proteases in the mechanisms underlying insect resistance to Bt toxins.

Highlights

Bacillus thuringiensis (Bt) are gram-positive entomopathogenic bacteria most widely used as a biopesticide worldwide, and transgenic crops expressing insecticidal toxins produced by these bacteria have been planted in 104 million hectares globally in 2018, which has a central role in pest control and global food security [1,2]
Differential expression of midgut trypsin-like serine protease (Tryp_SP) genes was identified in the Cry1Ac-resistant strain SZ-R in contrast to the susceptible P. xylostella strain
The role and function of the insect proteases present in midgut tissue in mediating Bt resistance has been analyzed in different P. xylostella resistant strains

Summary

Introduction

Bacillus thuringiensis (Bt) are gram-positive entomopathogenic bacteria most widely used as a biopesticide worldwide, and transgenic crops expressing insecticidal toxins produced by these bacteria (transgenic Bt crops) have been planted in 104 million hectares globally in 2018, which has a central role in pest control and global food security [1,2]. Cry protoxins are solubilized in the alkaline environment of the midgut and are further processed into activated toxins by midgut proteases when ingested by susceptible larvae [5]. Activated toxins interact with specific midgut receptor proteins, such as aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin (CAD) and ABC transporters, located in the brush border membrane (BBM) of the midgut epithelium cells from the larvae [6,7] Receptor binding leads to the formation of lytic pores in the membrane that burst cells and kill the insects [8]. It has been shown that protoxins bind to specific receptors, and they are activated by midgut proteases inducing toxin oligomerization and pore-formation [8,9]. Whether protoxins are activated before or after receptor binding is an important step, transforming the 130 kDa protoxin into a 55–65 kDa activated toxin [4,7]

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Conclusion